Development and validation of a quadruplex qPCR assay for simultaneous detection of GPV, GoCV, TMUV, and GAstV-Ⅱ in geese

Co-infections with multiple viruses are common in geese, complicating clinical diagnosis. Here, we developed a quadruplex TaqMan-probe based qPCR method for the simultaneous detection of Goose parvovirus (GPV), Goose circovirus (GoCV), Tembusu virus (TMUV), and Goose astrovirus Ⅱ (GAstV-Ⅱ). Specific primers and hydrolysis probes were designed targeting the following conserved regions: the VP3 gene of GPV, the Rep gene of GoCV, the E gene of TMUV, and the ORF2 gene of GAstV-Ⅱ, respectively. No cross-reactivity was observed with other common avian pathogens, including Goose paramyxovirus (GPMV), Fowl adenovirus (FADV), Newcastle disease virus (NDV, genotype VII ), Escherichia coli (E. coli), Pasteurella multocida (P. multocida). The limits of detection (LOD) for GPV, GoCV, TMUV, and GAstV-Ⅱ were 100, 100, 10, and 100 copies/µL, respectively. Intra- and inter-assay coefficients of variation ranged from 0.03% to 3.3%. Within the linear range of 10¹⁰ to 10⁴ copies/µL, the R² values for each virus were 0.9984, 0.9988, 0.9972, and 0.9978, respectively. The amplification efficiencies were 99.66% for GPV, 99.29% for GoCV, 100.41% for TMUV, and 104.48% for GAstV-Ⅱ. A total of 72 clinical samples collected from diseased goose between 2023 and 2024 were tested using the established method. The detection rates were 23.61% (17/72) for GPV, 13.89% (10/72) for GoCV, 9.72% (7/72) for TMUV, and 52.78% (38/72) for GAstV-Ⅱ. The co-infection rates for two pathogens ranged from 1.39% to 9.72%, while the rate of triple-pathogen mixed infection was 2.78%. In conclusion, a efficient and specific quadruplex qPCR method was successfully developed for the simultaneous detection of GPV, GoCV, TMUV, and GAstV-Ⅱ. This method provides a reliable technical tool for the differential diagnosis and epidemiological investigation of these four goose-origin viruses.